Peptic ulcer disease exists in two forms, duodenal ulcers and gastric ulcers. Central to the cause of duodenal ulcers, is the production of excess stomach acid and pepsin and a rapid gastric emptying time. This results in an increase in duodenal exposure to secreted acid and enzymes, and in mucosal damage.
The second form of the disorder, gastric ulcer disease, is caused by increased stomach acid and a breakdown of the complex stomach defenses that normally protect the gastric mucosa from acid damage. Although the two conditions have different etiologies, both benefit from a reduction in gastric acid secretion.
Because excess stomach acid is a central cause of ulcers, antacid preparations are commonly used as one method of treatment. This method merely neutralizes stomach acid after it is produced. Consequently, large quantities of antacids must be consumed on an ongoing basis to neutralize acid which is continually produced in the stomach. Antacids do not cure the disease because they do not affect the source of acid production.
Gastric acid is produced in a specialized stomach cell, the parietal cell. Parietal cells can be stimulated to secrete acid by acetylcholine, histamine and gastrin, upon the binding of each of these compounds with specific receptors on the surface of the cell. Of these the most potent stimulator of acid secretion is the peptide hormone gastrin.
Current approaches to the control and cure of peptic ulcers center upon devising drugs that inhibit the ability of one or more of these compounds to stimulate acid production or secretion. The most effective group of drugs approved for sale are the H2 antagonists (e.g. Tagamet.RTM. and Zantac.RTM.) which block the histamine H2 receptors on gastric parietal cells and inhibit acid secretion. These drugs, however, require relatively large doses on a daily basis and may induce several undesirable side effects. In cases where H2 antagonists have cured ulcers, relapses occur in almost 100% of cured individuals within a year of discontinuation of treatment. Other drugs have also exhibited problems, including low efficacy and unacceptable levels of toxicity. In the case the peptide hormone gastrin, no usable chemical antagonists have been identified.
Gastrin has several important functions in the gastrointestinal tract, the two most important being stimulation of acid secretion and stimulation of the growth of cells in the gastrointestinal tract. The hormone exists in at least two molecular forms, heptadecagastrin ("G17") and tetratriacontagastrin ("G34") named according to the number of amino acid ("AA's") residues in each molecule G34 and G17 are identical in structure at the carboxy terminus, which is the binding site of the hormones with receptors G17 constitutes the 17 carboxy terminal ( C-terminal") end residues of G34. G34 consists of the 17 C-Terminal end residues which comprise G17 and an additional different amino acid sequence of 17 amino terminal ("N-terminal") residues. When G34 is split by trypsin a G17 subunit and a non-hormonal 17 amino acid subunit results. Though G17 can be obtained by trypsin cleavage of G34, it is currently believed that this is not the normal route for the in vivo generation of G17. It appears that, in vivo, each form is generated separately from its own prohormone.
Although G17 and G34 are thought to be equipotent on a molar basis as stimulators of acid release, G34 is most probably responsible for the stimulation of growth of the gastrointestinal mucosa and the maintenance of the basal acidity of the stomach. G34 is the principal form present during interdigestive periods. G34 has a serum half life approximately six times as long as G17 (40 minutes versus 6 minutes) and is produced in both the stomach and the duodenum. Alternatively, G17 is the primary stimulator of meal induced gastric acid secretion. G17 is 1500 times more potent than histamine and makes up 90% of the antral (stomach) gastrin. G17 accounts for roughly 60% of the gastrin-mediated acid release.
The prior art in the area of gastrin immunology mainly concerns the induction of antibodies useful for identifying anatomic sites containing or producing gastrin G17 or G34 in laboratory animals; see Sugano, K., et al., 1985, "Identification and characterization of glycine-extended post translational processing intermediates of progastrin in porcine stomach", J. of Biological Chemistry 250: 11724-11729; Vaillant, C., et al., 1979, Cellular origins of different forms of gastrin: The specific immunocytochemical localization of related peptides. J. Histochem Cytochem 27:932-935; Larsson, L. I. et al., 1977, "Characterization of antral gastrin cells with region-specific antisera". J. Histochem. Cytochem 25: 1317-1321. The antisera reported in these publications contained antibodies of numerous specificities, for a variety of antigenic epitopes on gastrin molecules.
Attempts to control gastrin levels by anti-gastrin antibodies induced by active immunization or passive administration of preformed antibodies such as those reported in Jaffe, B. M., et al., 1971, "Gastrin resistance following immunizations to the C-terminal tetrapeptide amide of gastrin, Surgery 69: 232-238; Jaffe, B. M., et al., 1970, "Inhibition of endogenous gastrin activity by antibodies to the carboxyl terminal tetrapeptide amide of gastrin", Gastroenterology 58: 151-156; Jaffe et al., 1969, "Inhibition of endogenous gastrin activity by incubation with antibodies to the C-terminal tetrapeptide of gastrin. Surgery 65: 5633-639 are different from the present invention in that the immunogen used was derived from the carboxyl terminal tetrapeptide amino acid sequence common to G17, G34, and to another important hormone, cholecystokinin ("CCK"). The immunogen of Jaffe et al. is thus of no practical value as an anti-gastrin vaccine component; on the contrary, it would produce a deleterious state in which all gastrin activity and other hormone function of G17, G34, together with CCK, would be blocked and eliminated by immunization.
This invention provides a novel immunological approach to the control and regulation of peptic ulcers. According to the invention, antibodies are induced in the patient by passive or active immunization with immunogens that selectively target specific forms of gastrin.
Since the different forms of gastrin vary in function, it is necessary to selectively neutralize specific forms of gastrin to control specific functions. To regulate gastrin mediated secretion of stomach acid following meals (the principal source of excess stomach acid relating to ulcers), an immunogen must specifically target G17.
In order to selectively neutralize G17, one or more antigenic epitopes on G17 that are not found on G34 or cholecystokinin which exhibits carboxy terminal homology with gastrin must be identified. As discussed above even though the C-terminus of G17 and G34 are identical the N-terminus of G17 is very different from that of G34. This results in antigenic epitopes that are unique to G17 and can be separately targeted. We have identified and mapped such a unique epitope on G17. The present invention concerns immunogens comprising this unique epitope. These immunogens result in high levels of anti-G17 antibodies that do not crossreact with G34 and block some or all of G17 stimulation of gastric acid secretion while still allowing G34 and CCK, which share with G17 a common receptor, to carry out their physiologic function. The regulation of acid secretions can also involve the neutralization of G34; we have also identified and mapped unique epitopes on G34 that are not found on G17 or CCK.
Our immunoneutralizing approach has several attractive advantages over current treatments for peptic ulcer. One of these advantages is the overcoming of the major problem of patient compliance since a daily dose of a drug is not required. This invention treats ulcers b preventing the release of excess stomach acid, unlike antacids that neutralize secreted acid. By administering our synthetic peptide as an immunogen, the frequency and quantity of treatment administration is decreased, while at the same time long-lasting control of acid production, reduced side effects and easier patient administration are provided. Unlike conventional anti-ulcer drugs, antibodies generated by the peptide immunogens are very specific to their target. They do not cross the blood-brain barrier, and their use avoids certain complications encountered with drugs. In addition, unlike this invention, agonists or antagonists of G17 cannot be used to specifically control ulcers because such compounds would also occupy the receptors for G34 and CCK, which have identical receptor binding sites with G17.
The immunogens against one form of gastrin, "little gastrin", or G17, are constructed to produce an anti-gastrin vaccine component that will induce a selective and specific antibody response to G17 in the vaccinated human or other vertebrate, but not to G34 or CCK. This selective immunization to produce G17 specific antibodies is crucial to avoid producing antibodies specific for or cross reactive with G34, which might during the treatment of a specific condition induce undesirable side effects by blocking G34 physiologic functions. The antibodies resulting from the immunization with such immunogens target the chemical structure of G17 which is antigenically and immunogenically unique from the structure of G34. The amino acid residues beginning from the amino terminus (amino acid residue number one) of G17 and extending up to and including amino acid residue number 12 having the sequence pyro-Glu-Gly-Pro-Trp-Leu-Glu-Glu-Glu-Glu-Glu-Ala-Tyr, are used to prepare the immunogens of the invention. For simplicity this sequence can be written based upon the international code for amino acids as pyro-E-G-P-W-L-E-E-E-E-E-A-Y. The immunogens may contain a part or all of this sequence. For example, immunogens may be prepared by using the following specific peptide sequences: pGlu-Gly-Pro-Trp-Leu-Glu-Glu-Glu-, pGlu-Gly-Pro-Trp-Leu-Glu-Glu-, pGlu-Gly-Pro-Trp-Leu-Glu-, pGlu-Gly-Pro-Trp-Leu- and pGlu-Gly-Pro-Trp-. The last 5 carboxyterminal end amino acids of the G17 chemical structure (residues 13-17) are preferably not used, because this sequence is a common antigenic sequence between G17, G34, and at least one other hormone, cholecystokinin (CCK). Fragments, extensions, or other subsets of the natural hormone and of this 12 amino acid sequence of G17 may be used. One or more other amino acids may also be substituted for those of the natural sequence, so that increased or decreased binding capacity, specificity and/or titer of the antibody response against G17 may be induced in the vaccinated host by the immunogen.
The immunogens of the invention may be produced through synthetic or other processes commonly used in the art including standard peptide synthesis technologies; methods employing recombinant DNA and associated technologies; antigen mimicking methods including antibody-internal image technology and any other related methodologies that produce a structure that immunologically resembles the antigenic structures of G17 (mimotopes) and others.
In other embodiments of the invention the use of preformed G17 specific polyclonal and/or monoclonal antibodies and their derivatives or fragments produced by immunization, hybridoma, recombinant DNA or other technologies as a method of passive immunization for the control of gastric acid secretion stimulated by G17 may be used.
The present invention also provides immunogens against a second form of gastrin, "big gastrin" or G34. These immunogens are used to produce an anti-gastrin vaccine component that may be useful for the treatment or prevention of other gastrointestinal diseases and that will induce a selective and specific antibody response to G34 (but not to G17 or CCK) in the vaccinated human or other vertebrate. This selective immunization to produce G34 specific antibodies is crucial to avoid producing antibodies specific for or cross reactive with G17.
The G34 immunogens specifically target chemical structures of G34 which are antigenically and immunogenically unique from the structure of G17. The chemical structures of G34 utilized in this invention include, but are not limited to, the amino acid residues beginning from the amino terminus (amino acid residue number one) of G34 and extending up to and including amino acid residue number 22. The sequence of this peptide is pyro-Glu-Leu-Gly-Pro-Gln-Gly-Pro-Pro-His-Leu-Val-Ala-Asp-Pro-Ser-Lys-Lys-G ln-Pro-Trp-Lev. Based upon the international code for amino acids, this sequence is pyro-E-L-G-P-Q-G-P-P-H-L-V-A-D-P-S-K-K-Q-G-P-W-L-. The G34 immunogens may contain part or all of this sequence. The sequence of the last 12 amino acids of the G34 chemical structure (residues 23-34) are preferably not used in this invention because this sequence is a common antigenic sequence between G17 and G34. The sequence of amino acids are also not used since the sequence 29-34 has common antigenic sites with cholecystokinin. It is contemplated that the use of any fragments, extensions, or other subsets of the natural hormone and of this 22 amino acid sequence may be used as immunogens. One or more other amino acids may also be substituted for those of the usual natural sequence, so that increased or decreased binding capacity, specificity and/or titer of the antibody response against G34 may be induced in the vaccinated host by the immunogen.
The G34 immunogen may be produced by any process commonly used in the art including, for example, standard peptide synthesis technologies; methods employing recombinant DNA and associated technologies; antigen mimicking methods including antibody-internal image technology and any other related methodologies that produce a structure that immunologically resembles the antigenic structures of G34 (mimotopes).
Preformed G34 specific monoclonal antibodies and their derivatives or fragments produced by hybridoma, recombinant DNA or other technologies may also be used as a method of passive immunization for the control o gastric acid secretion stimulated by G34.
It may be desirable in some applications to immunize against both G17 and G34. In this embodiment G17 and G34 immunogens are used in combination including optionally an immunogen with an epitope common to G17 and G34, so that antibodies against both G17 and G34 are induced by the immunized host. The immunogens of this invention are therefore useful for more than just the treatment or prevention of ulcers. The immunogens may be used to treat any disease in which the gastrin stimulated secretion of stomach acid is a factor.
For the G17 and/or the G34 epitopes of this invention to induce antibodies, it may be necessary to increase their immunogenicity by chemically coupling them to other molecules. Such molecules are termed "carriers". Any molecule capable of serving as a carrier may be used. Examples of carriers for this purpose include: diphtheria toxoid, tetanus toxoid, keyhole limpet hemocyanin, bovine serum albumin, etc. Fragments of these carriers, including single epitopes, may also be used. Any method of chemically coupling the epitopes to the carriers may be followed. A preferred method utilizes the bifunctional linking agent EMCS described in U.S. Pat. No. 4,302,386; Lee et al., 1981.
The epitopes can be alternatively rendered immunogenic by crosslinking a number of epitopes. For this purpose, it may be necessary to extend the molecule bearing the G17 or G34 epitope by the addition of selected compounds that provide structures through which the crosslinking will occur. These additions must not disrupt the structure of the gastrin epitope, because the capacity to induce anti-gastrin antibodies would be lost. For example, to the carboxy terminal end of the G17 epitope pyro-E-G-P-W-M-E-E is added the amino acid sequence K-R-P-P-P-P-K, to give pyro-E-G-P-W-M-E-E-K-R-P-P-P-P-K. This molecule is then polymerized with glutaraldehyde, which crosslinks the lysine K residues, to form the crosslinked immunogen. This crosslinked immunogen should induce specific antibodies against G17.
In addition, the carrier and crosslinking methods may be used in combination. Thus, carrier epitopes that enhance immunogenicity and epitopes unique to G17 and/or G34 are integrated into the same polymer. For example, one or more carrier epitopes from Diphtheria toxoid may be crosslinked in combination with the G17 epitope pyro-E-G-P-W-L-E-E-K-R-P-P-P-P-K by the glutaraldehyde approach to yield an immunogenic copolymer. In a second example, a carrier epitope can be built into the molecule that contains the G17 epitope, then this larger molecule is crosslinked with glutaraldehyde. As a third example, the G17 epitope bearing molecule pyro-E-G-P-W-L-E-E-K-R-P-P-P-P-K may be crosslinked with a crosslinking agent that itself contains a carrier epitope.
The means by which anti-gastrin antibodies prevent acid release has not been thoroughly established. Without being bound by theory, we believe that the acid suppressive effect of our immunogen is due to the binding of anti-gastrin antibodies to gastrin (G17 and/or G34) in the blood, and thereby preventing the binding of gastrin to its physiological receptors on the surfaces of parietal cells. Thus, gastrin is prevented from signaling parietal cells to secrete acid into the stomach.
Administration of these immunogens, compositions containing them, or pharmaceutically acceptable and immunologically effective derivatives thereof, may be via any of the conventionally accepted modes of administration of agents which exhibit immunogenicity. These include oral or parenteral administration.
The compositions used in these vaccines may be in a variety of forms. These include, for example, solid, semisolid and liquid dosage forms, such as powders, liquid solutions or suspensions, suppositories, injectable and infusible solutions. The preferred form depends on the intended mode of administration and therapeutic applications.
The compositions also will preferably include conventional pharmaceutically acceptable vehicles or carriers and may include other medicinal agents, carriers, adjuvants, excipients, etc., e.g., human serum albumin or plasma preparations. Preferably, the compositions of the invention are in the form of a unit dose. The amount of active compound administered as a vaccination or as a medicament at one time, or over a period of time, will depend on the subject being treated, the manner and form of administration, and the judgment of the treating physician. However, an effective dose may be in the range of from about 1 ug to about 10 mg of the immunogen of this invention, preferably about 100 (micrograms "ug") to about 2 mg; it being recognized that lower and higher doses may also be useful.
The examples illustrate specific embodiments of the invention. It should be understood that these examples are for illustrative purposes only, and are not to be construed as limiting this invention in any manner.